Virtually all eukaryotic cells transport organelles and a subset of mRNA molecules to specific destinations within the cytoplasm. Such transport generates asymmetric distributions of macromolecules, which is critical for establishing the body plan during early development and for established cell polarity and specialized intracellular domains. Hence deciphering the mechanism of intracellular transport is fundamental for understanding many basic biological processes. Medical implications include possibly developing intracellular drug delivery systems and treating neurodegenerative diseases (some of which may be due to impaired transport). Motor proteins (kinesin, myosin, and dynein) and cytoskeletal elements (microtubules and actin filaments) are essential molecules for intracellular transport. In addition, the motor protein must be attached to the correct cargo by linker molecules. This grant proposes to investigate how motor proteins interact with two types of cargo, membrane organelles and mRNA, and to explore how these interactions are regulated. Membrane transport ha been a focus of this grant for the past ten years. The investigation of mRNA localization and interaction with the cytoskeleton represents a new and relatively unexplored problem to which this laboratory~s expertise in biochemistry and in vitro motility assays could be applied. The specific goals of this grant are to: 1) Identify a mitotically-activated dynein kinase and determine the role of dynein phosphorylation in membrane and chromosome attachment. 2) investigate the structural requirements of dynein binding to membranes by systematically mapping domains of the dynein heavy chain that are required for attaching the motor to membranes. 3) isolate, biochemically characterize and develop in vitro motility and/or cytoskeletal binding assays for cytoplasmic, localized mRNA particles. Two systems have been chosen, where biochemical work can be augmented by genetic studies from other labs. In Drosophila oocytes, a particle containing exuperantia (exu), a protein that is involved in bicoid mRNA localization and moves along microtubules, will be isolated and characterized. In S. cerevisiae, localization of Ash-1 mRNA to the bud site will be explored by biochemical techniques. 4) Purify and identify and function of a protein that represses translation of the Vg1 localized mRNA. The goal of this specific aim is to understand how translation of a localized mRNA is coordinated with its arrival at its final target destination. Overall, the experiments in this proposal are designed to further our molecular understanding of how cells asymmetrically position components with their cytosplasm.